An optical control system is described, which employs a series of optical emitters and detectors arranged about a touch area. The emitters are driven by a series of orthogonal functions, and the system is operable to correlate the signals received at the detectors with the modulation functions to determine the amount of energy received by the detectors from each of the emitters. This system means that all or many emitters can be modulated simultaneously, so the scanning process is accelerated. Also, the energy estimates arrived at by correlation with the modulating functions are largely unaffected by external interference signals as well by each other. An additional benefit of the present invention is that the correlation processing makes use of many detector samples and tends to yield results which have higher resolution than the sample values themselves, due to the averaging of noise over the sample set.

1. Field of the Invention
This invention relates to optical control systems and devices, for example, touch sensitive displays and touch sensitive overlays for displays.
2. Description of the Related Art
In a traditional optical touch overlay, optical emitters and detectors are arranged along opposing edges of a rectangular touch sensitive area. An orthogonal grid is formed by the optical beams between the emitters and the corresponding detectors over the touch sensitive area. The beams are scanned in sequence to determine if the path loss indicates that there is an object (such as a finger or a stylus) interrupting or modulating any of the beams.
The touch sensitive area may be an optically transparent planar waveguide through which the beams pass by total internal reflection, or a surface over which the beams pass in close proximity to the surface and substantially parallel to the surface.
In the case of a waveguide, the material used can be a transparent sheet of plastic or glass. An object such as a finger or a stylus coming into contact with the transparent waveguide will have a higher refractive index than the air normally surrounding the waveguide. The increase in refractive index will disrupt total internal reflection of light energy at the interface between the waveguide and the touching object, causing increased light leakage from the waveguide over the area of contact. This leakage attenuates any beams passing through the position of the touch event. Correspondingly, the removal of the touching object will cause the attenuation of the passing beams to be reduced as will be detectable at the output of the associated detectors.
In the present specification the term "light" includes IR and UV radiation, and the term "optical" is to be interpreted accordingly.
When the touch sensitive area is a surface over which the beams pass, an object which is not optically transparent at the wavelength in use will attenuate or interrupt the beams passing through the object location.
This is illustrated in FIG. 1, where an opaque object 10 at position T attenuates a beam 12 between an emitter 14 on the left side of a touch sensitive area 16 and a detector 18 on the right side of the touch area 16. The opaque object 10 also interrupts a beam 20 between orthogonal axis emitters 22 and the corresponding detectors 24.
Scanning logic circuitry associated with the emitters and detectors can determine the intersection point of one or more interrupted beam paths and deduce the position of the interrupting object. The object must be large enough to detectably attenuate at least one beam in one axis and at least one beam in the orthogonal axis.
Such a panel traversed by optical paths can, in addition to the detection of touch events, detect the operation of mechanical control devices arranged along the optical paths. For example, a mechanical button control may be inserted along an optical path whereby the button control presents a minimal attenuation to optical energy when the button actuator is in its resting position, but introduces significant optical attenuation when the button actuator is pressed. This may be accomplished by introducing an opaque vane into the optical path when the button is pressed. Various mechanical designs for control devices may be used to modulate the optical energy passing along the optical paths such that the status of such control devices can be detected by processing the signals from the optical detectors.
The use of amplitude modulation of optical emitter outputs in optical touch and related optical control systems is well known. Typically, a specific frequency of modulation is chosen which can readily be separated from unwanted signals at the detector output by narrowband filtering. This modulating signal can be applied to an emitter by using it to modulate the current flowing through the emitter, which is usually be a current controlled device such as an LED.
However, optical emitter and detector response times (often tens of microseconds for detectors) and the need to be distant in frequency from the harmonics of mains-powered light sources limit the range of usable frequencies in low cost applications, so most devices using airborne optical transmission have optical emitters which are amplitude modulated by a fixed frequency in the 30 kHz to 80 kHz range.
Therefore, any device using optical detectors may receive external interference originating from other devices with emitters radiating light of a similar wavelength which is modulated by a similar range of frequencies.
Optical scanning devices normally activate emitters (modulated or unmodulated) in a sequential manner, so that the signal at one or more active detectors can be associated with a known source emitter. This prolongs the scanning process, particularly if modulation needs to be detected for each emitter signal to be determined reliably.
The invention provides an optical control system comprising:
a plurality of optical emitters and a plurality of optical detectors defining therebetween an area of optical paths whereby the transmission of energy between an emitter and a detector may be modulated by an optical interaction occurring in the vicinity of said area, wherein at least one of said detectors is positioned to receive optical energy from more than one of said optical emitters;
drive circuitry for driving a plurality of said optical emitters simultaneously according to a modulation scheme in which each emitter is modulated with a different modulation function, said functions being orthogonal relative to one another;
correlation means associated with said at least one detector for receiving an output therefrom and correlating said output with at least those modulation functions driving those emitters which said at least one detector is positioned to receive optical energy from, to thereby determine the amount of energy incident on said at least one detector from each of said emitters.
In the present invention, functions which are orthogonal to one another are used to amplitude modulate the emitter devices, with one orthogonal function allocated to each emitter. Preferably, each modulating function has a broad frequency spectrum and a strongly non-periodic nature in the time domain so that it will not correlate well with interference signals, which are typically periodic.
In the present specification the term "orthogonal" refers to any two chosen functions from a family of "orthogonal" functions having zero cross-correlation.
Preferably, each modulating function has just two values over time so that digital control of the emitter may be used for simplification of the associated circuitry and processing. The depth modulation applied to the emitter may be any value from greater than 0% up to 100%.
There exist families of complex functions which are orthogonal to each other. If each emitter in a group of emitters is allocated a function from an orthogonal family of functions, the emitters may be simultaneously modulated by their respective functions but the energy from each emitter arriving at a given detector will still be measurable separately. By correlating the output from a detector which receives the composite energy from at least one emitter with each of the modulation functions in use, the resulting correlation values are accurate estimates of the energy received from the emitter associated with each modulation function.
FIG. 2 shows the hardware sections of an optical control system. The microprocessor 26 writes emitter modulation patterns to the emitter register 28 under software control. The sample values for at least one of the emitters 30 are written at the start of every sample interval. The beams 32 travel across the touch sensitive area 34 and, if not interrupted by an opaque object, arrive at a detector 36. The analogue multiplexer 38 selects the analogue output of one detector 36 as directed by the value in register 40 and routes it to the analogue-to-digital converter 42 for conversion into numeric form for processing by the microprocessor 26. The analogue multiplexer 38 may select the outputs of more than one detector during a single sample interval.
The touch sensitive area 34 can be an optically transparent planar waveguide through which the beams pass by total internal reflection, or a surface over which the beams pass in close proximity to the surface and substantially parallel to the surface, to give two examples. The emitters and detectors are shown as two simple arrays on opposite edges, but in practice, emitters and detectors may be arranged around the entire perimeter of the surface (e.g. all four edges of a rectangular surface) to maximise the resolution of, the touch sensitive system.
The current drawn by the emitters 30 when they are radiating will usually be substantial and it is advantageous to minimize the time for which the emitters 30 are active. Therefore, it may be efficient in a device with current limitations for the microprocessor 26 to update the emitter register 28 at the start of every sample period and to control the analogue multiplexer 38 by means of register 40 such that the signals from more than one detector 36 are routed to the analogue-to-digital converter 42 in rapid succession and the resulting sample values written to RAM 44 for processing later. Before the first analogue-to-digital conversion can be performed by the converter 42 in a given sample interval, there may need to be a pause corresponding to at least the response times of the emitters 30 and the detectors 36. When the sample and store process is complete for all of the detectors 36, the sample period can end and the next sample period can commence with the updating of the emitter register 28. At least one digital sample of the output of the detectors 36 is required for every update of the emitter register 28. When there has been a sample interval with an emitter update and sample acquisition for each bit of the modulating functions, the emitters 30 can all be rendered inactive by the controlling software and the sample values in the RAM 44 can be correlated with the modulating functions.
The use or a family of orthogonal modulation functions in the present invention means that all or many emitters can be modulated simultaneously, so the scanning process is accelerated. Also, the energy estimates arrived at by correlation with the modulating functions are largely unaffected by external interference signals as well by each other. An additional benefit of the present invention is that the correlation processing makes use of many detector samples and tends to yield results which have higher resolution than the sample values themselves, due to the averaging of noise over the sample set.
A given family of orthogonal functions will have a minimum period over which the member functions of that family are fully orthogonal. That minimum period will typically be a number of samples which is a power of two. There will be more functions in an orthogonal family when the number of samples is increased (i.e. a longer code length is used), permitting more emitters to be simultaneously active. However, long codes will also incur a longer scanning time for a given modulation rate. Ideally, the shortest code length which provides an orthogonal modulation function for each emitter in a group (where a group might be all of the emitters in a system or might be the emitters relating to an X or Y axis) would be used.
In selecting modulation functions from a family of orthogonal functions, some may be more desirable than others. For example, the families of Walsh functions are families of mainly complex orthogonal functions where each has only two values over time, but some members are purely periodic i.e. they are square waves with a constant period. The non-periodic functions are to be preferred since the functions used preferably have the broad spectrum mentioned above. However, this constraint reduces the number of family members which can be used for a given code length.
FIG. 3 shows an example family of orthogonal functions with a code length of 16 samples. The cross correlation of any of these functions with any other function in the family is zero over the code length.
Function numbers 3, 4, 7, 8, 9, 10, 11 and 12 have been marked with an asterisk to indicate that these are preferable to the other family members because they are non-periodic. (Even so, functions 4 and 12 are still not ideal, as they have only one phase change and are therefore substantially periodic.)
In an additional aspect of the invention, the emitter on and off periods are varied in a complex manner. This permits the use of periodic modulation functions. The sampling of the detectors must be varied similarly so that the sample values presented to the correlation processing will be essentially unaffected by the timing variations. The timing variations can be derived from a complex function such as a pseudorandom sequence and the variations are applied globally so that all emitters change state at the same time and all detectors are sampled at approximately the same time (the use of a multiplexed analogue-to-digital converter means that they cannot be sampled truly simultaneously)
An external interference signal is likely to be modulated in a periodic manner and FIG. 4 shows that it is possible that it will correlate with a periodic emitter modulation function of similar frequency and phase. Function 13 in FIG. 4 is the modulation function 13 from the family of functions shown in FIG. 3. Function 13 of the example family is periodic and the transition between samples is marked with an arrow. It will be evident from the diagram that the external periodic interference signal shown is of similar frequency and phase to function 13 and would correlate strongly with function 13.
In accordance with this additional aspect of the present invention, function 13′ in FIG. 4 has the same sequence of values as function 13, but the timing between the application of the function values to the associated emitter is varied and inconsistent. The transition between samples is again marked with an arrow. The detector sampling is varied in the same way so the correlation with the emitter modulation functions will be unchanged while the correlation with external periodic interference signals, which are similar in frequency to the nominal frequency of the modulating function, will be significantly reduced.
This timing variation can be applied even if only non-periodic emitter modulation functions are being used, so that the spectral components of the emitter modulation function are spread in frequency around the harmonics of the nominal modulation frequency. This will increase the rejection of typical interference signals, particularly when short code lengths are used.
In another aspect of the invention, members of a family of orthogonal functions may be chosen such that the number of emitters active at any stage during the length of the codes is limited to a maximum number. Without applying this criterion, many combinations of orthogonal functions will result in at least one step in the code length where all or all but one of the emitters are active.
If the code length is large, the number of available orthogonal functions may be large—tens of thousands for a 32-bit code length—and iterative methods can be used to select functions with the minimum number of simultaneous '1' or '0' values (whichever value corresponds to an emitter being activated).
FIG. 5 lists a set of values (in decimal and binary) for modulating 16 emitters with every bit in the binary representation word assigned to an emitter and with modulating functions of 32 bits in length. A given bit position in each of the 32 words represents the values of one orthogonal function. The set of values listed is suitable for driving emitters where an emitter is activated by a '1' value in the corresponding bit of each 16-bit word. None of the functions listed are periodic.
Thus, for each emitter, the sequence of values used to drive the emitter through a series of 32 "on" or "off" states is found by reading down the columns. Each row is a 16 bit word used to drive the 16 different emitters to the on or off state represented by a different position. So, for example, the first word (decimal 19418=binary 0100101111011010) is written to the register at the start of the first sample interval and determines that each of the first, third, fourth, sixth, eleventh, fourteenth and sixteenth emitters is driven to an "off" state, while the remaining emitters are driven to the "on" state during this first sample interval. The emitter states for the second sample interval are determined by the second row (decimal 40557=binary 1001111001101101), switching the first emitter on, the second emitter off, leaving the third emitter on, and so on.
The functions used to generate the list shown in FIG. 5 have been iteratively selected to minimise the number of emitters active at any time. In the case of the functions listed, there is no word which would activate more than 11 emitters at once. An advantage of this aspect of the invention is that it reduces the dynamic range of the light levels received at a detector. This can be translated into an improved signal-to-noise ratio in the signal acquired by the A/D converter used to digitize analogue detector outputs. An additional advantage of using functions selected in this way is reduced peak current demand caused by the relatively high operating current drawn by the emitters and reduced variation in current demand (power line noise) over the code length.
In some applications, it may also be advantageous to select functions where the minimum number of emitters that would be simultaneously active is large, so that the variation in light level at a detector and the variation in current consumption is kept to a minimal value. With the example functions shown in FIG. 5, there are never fewer than 3 emitters active at a time, so the range of light levels at the detectors will vary between the output of 3 emitters and the output 11 emitters, assuming none of the beams are interrupted. This may also be advantageous in other circumstances, such as where the detectors are phototransistors, a minimum illumination level biases the phototransistors into a relatively linear part of their characteristic curve.
In yet another aspect of the present invention, more than the requisite single sample of a detector output is acquired during a sample interval. At least one of the additional samples taken is acquired with the emitters inactive.
The taking of additional samples when the emitters are inactive is well known for the purposes of ambient light compensation. However, it is also advantageous to take such samples with a precise timing relationship to the modulation of the emitters to minimize the effects of rapidly modulated interference as well as ambient light. This can be accomplished by acquiring samples of the detector output before and after the emitters are active for a given step of their emitter functions.
FIG. 6a shows an example of a sampling sequence in accordance with this additional aspect of the present invention. The timescale shown covers one bit period in the length of a code. First, the emitters are forced off and a sample is taken when the detectors have settled at the resulting light level. The sample value acquired will be determined by the amount of external light which is effectively unmodulated, such as sunlight, and the amount of modulated light, such as incandescent light, which is incident upon the given detector. A second sample is taken during the emitter activation period. It is during this time that the various functions allocated to each emitter specify whether the corresponding emitter should be activated or not for this bit period. FIG. 6b shows an example of a bit period where at least one of the emitter functions dictates that the associated emitter is to be activated. A third sample is taken after the emitter has been turned off after the emitter activation period.
If the 'before' and 'after' samples are acquired with a known timing relative to the 'during' sample (when the emitters are emitting or not as determined by the current phase of their corresponding modulation functions), an intermediate estimate of the 'during' sample with the emitters off can be interpolated. If the time between the 'before' and 'during' samples and the 'during' and 'after' samples is the same, the interpolation is simply the sum of the 'before' and 'after' sample values divided by 2. This interpolated value can be subtracted from the actual sample acquired during the emitter activation time to compensate for ambient light and interference of low frequency relative to the sample rate.
Preferably, the interval between all three samples is short relative to the modulation rate of typical interferers.
A useful value can be derived from the 'before' and 'after' samples when the difference between them is calculated. This gives an approximate measure of the noise and interference present at frequencies which are high relative to the sampling rate. This noise estimate can advantageously be used to generate alarms or inhibit the generation of scan data if the amount of noise and interference is excessive (meaning the scan data is of dubious quality).
By comparing the noise estimate with the results of the correlation process, the signal-to-noise ratio of the signal at a detector can be estimated for each emitter. This can be used to control the emitter power as described in our co-pending specification (PCT application claiming priority from Irish Patent Application S2008/0653).
When sampling rapidly and with fixed timing, the 'before' sample of one function bit period may be the 'after' sample of the previous bit period.
FIG. 7 is a flowchart showing one way of generating a set of modulation functions. A set of aperiodic sequences of the required length is generated and tested for mutual orthogonality. The number of ones and/or zeroes in each position is checked across the set of sequences. If a threshold has been set (e.g. no more than 11 ones in the same position in each of 16 sequences) then a comparison is made with this threshold, and additional candidate sequences are substituted until the threshold is met. If no threshold has been set, the process continues by discarding and replacing sequences until an optimal solution is reached.
Alternatively, one could generate families of mutually orthogonal sequences and, within each such family, select the required number of functions (e.g. 16 functions to drive a group of 16 emitters) based on the optimal solution in terms of highest minimum number of simultaneous "off" states and/or lowest maximum number of simultaneous "on" states. This could be repeated for several such families of mutually orthogonal functions up to and including exhaustively testing all such sequences of length N to find the optimal solution.
The invention is not limited to the embodiment described herein but can be amended or modified without departing from the scope of the present invention.

Optical control system with modulated emitters


A method of treating prostate cancer in a living mammal includes local administration of a composition that includes a therapeutically effective concentration of collagenase. In one embodiment, a method of treating prostate cancer in a living mammal includes local administration of a composition that includes a therapeutically effective concentration of collagenase and at least one of a glycosidase, a protease, a nuclease, a lipase, an esterase, a plasminogen activator, a streptokinase, and combinations thereof. Preferably a glycosidase, such as, for example, hyaluronidase, is administered. Compositions used in methods for treating prostate cancer can also include or be administered with calcium ions, a nonionic surfactant, such as, for example, Triton(copyright) X-100, and an antibiotic, such as, for example, gentamicin. Another method of treating prostate cancer in a living mammal includes activating PSA in vivo by, for example, locally administering calcium ions.

The present invention is directed to a method for treating prostate cancer by administering collagenase alone or in combination with a glycosidase, a protease, a nuclease, a lipase, an esterase, a streptokinase, or a combination thereof. A typical embodiment includes administering a composition including a mixture of collagenase, hyaluronidase, a nonionic surfactant, an antibiotic, and calcium ions.
Approximately ten million American men are believed to have prostate cancer today. Although fewer than 3% of men with the disease die from it, prostate cancer still is the second most common cause of cancer death among men. The cancer usually is localized in the prostate, but in some cases, the cancer is not diagnosed until it has metastasized to the bone, kidneys, or the brain.
Yearly screening for the disease increases the likelihood of early detection, especially prior to the disease metastasizing. Such screening usually involves a digital rectal exam and a prostate-specific-antigen (PSA) blood test. Other screening methods include ultrasound imaging, radionucleid scan, and biopsy.
The PSA blood test has revolutionized the early diagnosis of prostate cancer and the effectiveness of treatment. PSA is a proteolytic enzyme in the family of serine proteases and one of the most abundant proteins in the prostate secretions. PSA is synthesized in the ductal epithelium and prostatic acini and is located within the cell in cytoplasmic granules and vesicles, rough endoplasmic reticulum, vacuoles, secretory granules, and lyosomal dense bodies. PSA is secreted into the lumina of the prostatic ducts where it becomes a component of seminal plasma. To reach blood serum, PSA diffuses from luminal cells through the epithelial basement membrane and prostatic stroma and either passes through the capillary basement membrane and epithelial cells or into the lymphatics. Once in the bloodstream, the majority of PSA forms complexes with xcex1-1-antichymotrypsin (PSA-ACT) and xcex1-2-macroglobulin, while small quantities remain free (free PSA). Free PSA levels are usually elevated in instances of prostate cancer.
Once prostate cancer is diagnosed, a suitable method of treatment must be determined and then administered. Current methods of treatment include radical prostatectomy, radiation, and hormonal suppression. To determine the appropriate method of treatment, factors such as the age of the patient and severity of the disease are often considered. The disease generally is more aggressive for younger patients. Any tumor greater than 0.5 cc is typically considered clinically significant. The preferred treatment for localized prostate cancer is radical prostatectomy. This treatment may result in death, incontinence, impotence, rectal injury, or pulmonary emboli.
Thus, it is desirable to provide improved methods of treatment for prostate cancer that reduce the likelihood of one or more of these unpleasant side effects. In particular, it is desirable to provide improved methods of treatment that reduce the likelihood of the treatment rendering the patient impotent.
The current invention is directed to a method of treating prostate cancer in a living mammal. In one aspect, the treatment is directed to activating PSA in vivo. PSA in or near the prostate can be activated by one of several methods, such as by local administration of calcium ion preferably in combination with collagenase and, optionally, another hydrolase such as hyaluronidase.
PSA is a proteolytic enzyme, which is one of the most abundant proteins in the prostate secretion. Activated PSA may degrade prostate cancerous tumors by, for example, interfering with the initial growth stage of a tumor and/or interfering with the angiogenesis stage. The initial stage of prostate cancer growth involves the tumor growing to about the size of a pea. The tumor cannot get any larger unless it can form its own blood vessels around and inside the tumor, a process known as angiogenesis. Angiogenesis involves sending chemical signals to surrounding blood vessels that erode the vessel walls, sending capillaries toward the tumor. By interfering with angiogenesis, the tumor cannot grow beyond its initial size. In one aspect, PSA may be activated by administering calcium ions.
In another aspect, a method of treating prostate cancer is directed to administering a composition that includes a therapeutically effective concentration of a collagenase. Collagenase may degrade prostate cancerous tumors by, for example, supporting connective tissue around and inside of the cancerous tumor including angiogenesis-generated capillaries.
In another aspect, a method of the invention is directed to local administration of a therapeutically effective concentration of collagenase in combination with a glycosidase, a protease, a nuclease, a lipase, an esterase, a streptokinase, or a combination thereof. Preferably a glycosidase, such as, for example, hyaluronidase, is administered with collagenase.
Compositions used in methods of the invention may also include or be administered in combination with calcium ions, a nonionic surfactant, such as, for example, Triton(copyright) X-100, and an antibiotic, such as, for example, gentamicin.
The present invention provides a method for treating prostate cancer. In accordance with the present invention, it has been found that compositions containing at least one hydrolytic enzyme can be used to treat prostate cancer. xe2x80x9cTreatmentxe2x80x9d and xe2x80x9ctreatingxe2x80x9d as used herein include preventing, inhibiting, curing, and alleviating prostate cancer or symptoms thereof and preventing or alleviating the metastasis of prostate cancer.
Alleviating prostate cancer includes degrading a prostate tumor, for example, breaking down the structural integrity or connective tissue of a prostate tumor, such that the tumor size is reduced when compared to the tumor size before treatment. Curing prostate cancer includes degrading a prostate tumor such that a tumor cannot be detected after treatment. The tumor may be reduced in size or become undetectable, for example, by atrophying from lack of blood supply or by being attacked or degraded by one or more components administered according to a method of the invention.
Alleviating metastasis of prostate cancer includes reducing the rate at which the prostate cancer spreads to other organs. Preventing metastasis of prostate cancer includes preventing the prostate cancer from spreading outside of the prostate.
A treatment according to the invention may be carried out by administering a therapeutically effective concentration of a composition including collagenase in an amount effective for alleviating, curing, inhibiting, or preventing prostate cancer or preventing or alleviating the metastasis of prostate cancer.
The composition including collagenase is locally administered to the prostate. Local administration includes delivering the composition into or near the prostate and/or cancerous tumor. Local administration further includes surrounding the prostate and/or cancerous tumor with the composition or applying the composition to the surface of the prostate and/or cancerous tumor. Cancerous tumor includes prostate cancer, cancerous cells, and the like.
Another treatment according to the invention may be carried out by activating PSA in vivo. Activating PSA in vivo includes activating PSA, typically the mammal""s endogenous PSA, in and/or near the prostate. PSA is a proteolytic enzyme that, upon being activated, can facilitate dissolution or degradation of a prostate tumor. In one embodiment, PSA can be activated by locally administering calcium ions to the prostate.
Although the methods of the invention are not meant to be limited to a single theory, one theory directed to how the methods of the invention treat prostate cancer will be described. In initial stages, a cancerous tumor grows to about the size of a pea. Through angiogenesis the tumor then grows larger (i.e., the tumor grows blood vessels around and inside itself by sending chemical signals to surrounding blood vessels to send capillaries toward the tumor). It is believed that the treatments of the invention interfere with this development.
PSA is a serine protease having a molecular weight of about 33 kilodaltons and is one of the most abundant proteins in the prostate secretion. It is believed that by activating PSA, PSA can partially or completely dissolve or degrade tumor cells in the prostate. This process can be particularly useful at the initial growth stage of a tumor as well as at the angiogenesis stage. PSA can be activated by, for example, administering calcium ions.
Collagenase is a metalloprotease. According to one theory, the activity of collagenase may be increased in the presence of a small amount of proteolytic enzymexe2x80x94i.e., the amount of proteolytic enzyme present should not be so high that the proteolytic enzyme digests the collagenase to such an extent that the collagenase becomes inactive. PSA secreted into the prostate may be sufficient to increase the activity of collagenase. By increasing the activity of collagenase, the activity of collagenase is raised from a level that may be ineffective for cleaving sufficient substrate (e.g., degrading connective tissue) to a level that may be effective for cleaving sufficient substrate. Then collagenase, in the presence of PSA, for example, can degrade collagen or connective tissue, tumor blood vessels, and/or basement membranes of the tumor cells. As a result, the tumor blood supply is reduced or eliminated, causing the tumor cells to die. This may also result in a person""s immune system further attacking the tumor and aiding the alleviation or cure of prostate cancer or the alleviation or prevention of metastasis of prostate cancer.
In methods of the invention, collagenase can also be administered in combination with other proteases, enzymes, or proteins suitable for increasing the activity of collagenase or for aiding in the degradation of a tumor. For example, collagenase can be administered in combination with a protease, a glycosidase, a nuclease, a lipase, an esterase, a streptokinase, or a combination thereof.
Glycosidases include any enzyme that catalyzes the hydrolysis of glycosidic linkages. Suitable examples include hyaluronidase, neuraminidase, amylase, and lysozyme. Preferably the glycosidase includes hyaluronidase.
Proteases include any enzyme that catalyzes the hydrolysis of one or more peptide bonds in a protein or peptide, such as, for example, carboxypeptidases, aminopeptidases, and endopeptidases. More specific suitable examples include elastase, trypsin, chymotrypsin, pronase, dispase, bromelin, clostripain, thermolysin, subtilisin, papain, chymopapain, fibrinolysin, serrathiopeptidase, pancreatin, cathepsin-G, plasminogen activator, and PMN leukocyte serine protease.
Nucleases include any enzyme that catalyzes the hydrolysis of ester linkages in nucleic acids, such as, for example, ribonuclease and deoxyribonuclease. Suitable examples include DNase I and RNase.
Esterases includes any enzyme that catalyzes the hydrolysis of an ester. A suitable example includes cholesterol esterase.
Lipases include any enzyme that catalyzes the hydrolysis of acylglycerol carboxylic esters. A suitable example includes phospholipase.
Streptokinases include proteins that form a complex with plasminogen that then catalyzes the activation of plasminogen to plasmin.
The compositions can also include or be administered in combination with calcium ions, a surfactant, and/or an antibiotic.
The components of the compositionxe2x80x94collagenase, additional protease, protein, or enzyme, calcium ions, surfactant, and antibioticxe2x80x94can be administered alone, sequentially, or preferably, combined with one another in the form of a liquid pharmaceutical unit dosage form suitable for local administration. The dose of the composition administered can vary over a wide range as can readily be determined by the clinician. The preferred dosage for obtaining the desired therapeutic objective can vary depending on the age of the patient, nature and severity of disease, potency of the composition, and route of administration.
Preferably, the compositions suitable for use in methods of the invention include collagenase and at least one of a glycosidase, preferably hyaluronidase; a protease, preferably trypsin, chymotrypsin, pronase, elastase, dispase, or fibrinolysin; or a nuclease, preferably DNase I. More preferably, the compositions include collagenase and a glycosidase, preferably hyaluronidase. Compositions suitable for use in methods of the invention have been described in U.S. Pat. No. 5,116,615 issued to Gokcen et al., incorporated herein by reference.
Collagenase
Bacterial collagenase, for example, Clostridium hystolyticum, EC 22.214.171.124, is a well-characterized, commercially-available enzyme that degrades collagen into small peptides by hydrolysis at several sites along the triple helix. Collagenase contains Zn+2 in its active site and requires Ca+2 for binding to its substrate and for achieving the conformation necessary for full catalytic activity.
Intravenous injections of collagenase have shown a very low degree of danger to experimental animals. In mice the IV LD-50 of crude collagenase has been shown to be 300 mg/kg body weight. Oral solutions of collagenase in water have been proven to be nontoxic at doses as high as 8,000 mg/kg body weight. The acute IV LD-50 in rats has been shown to be 1272 U/kg for collagenase.
Commercial preparations of bacterial collagenase often include small amounts of contaminating proteases, peptidases, mucopolysaccharidases, and glycosidases including:
clostripain, trypsin, and a caseinasexe2x80x94like aminopeptidase. Clostripain is the most abundant contaminating enzyme in crude collagenase preparations. Clostripain contains an essential SH group, is activated by cysteine, and is inhibited by sulfhydryl binding agents. Clostripain also has trypsin-like specificity. Crude collagenase preparations with small amounts of contaminating enzymes such as trypsin and clostripain are often more effective than highly purified preparations of collagenase, suggesting that a possible combined action of the multiple enzymes or proteases aids in the treatment of prostate cancer. These enzymes and/or proteases can help activate collagen and/or can help degrade the tumor by catalyzing the hydrolysis of bonds important to the growth or structural integrity of the tumor.
Crude collagenase preparations are especially useful for treatment when combined with other proteins and/or enzymes, such as, for example, a glycosidase, a protease, a nuclease, a lipase, an esterase, a streptokinase, or a combination thereof.
Glycosidases
Examples of glycosidases suitable for use in methods of the invention include hyaluronidase, neuraminidase, lysozyme, and amylase. Preferably the glycosidase is hyaluronidase.
Hyaluronidase (hyaluronate-4-glycanhydrolase) is a known enzyme that catalyzes the degradation of hyaluronic acid (an acidic mucopolysaccharide) into disaccharides, tetrasaccharides, or a mixture of both. Hyaluronidase derived from ovine testes (EC 220.127.116.11) is preferred.
In animals, the intravenous injections of 75,000 International Units of hyaluronidase results in no significant change in blood pressure, respiration, body temperature, or renal function. Hyaluronidase is typically not injected into areas of known infection.
Amylase includes enzymes, such as, for example, xcex1-amylase or xcex2-amylase, able to hydrolyze O-glucosyl linkages in starch, glycogen, and related polysaccharides.
Neuraminidase degrades 2,3-, 2,6-, and 2,8-glucosidic linkages joining terminal nonreducing N- and O-acylneuraminyl residues to galactose, N-acetylhexosamine, or N- or O-acylneuraminyl residues to galactose, N-acetylhexosamine, or N- or O-acylated neuraminyl residues in oligosaccharides, glycoproteins, or glycolipids.
Lysozyme hydrolyzes xcex2-1,4-linkages between N-acetylmuramic acid and 2-acetamido-2-deoxy-D-glucose residues in peptidoglycan heteropolymers. Lysozyme is identified by EC 3.21.17.
Proteases
Examples of proteases suitable for use in methods of the invention include elastase, trypsin, chymotrypsin, pronase, dispase, bromelin, clostripain, thermolysin, subtilisin, papain, chymopapain, fibrinolysin, serrathiopeptidase, pancreatin, cathepsin-G, plasminogen activator, and PMN leukocyte serine protease.
Elastase includes any protease that hydrolyzes elastin. One example is pancreatic elastase identified by EC 22.214.171.124. Pancreatic elastase catalyzes the hydrolysis of proteins, including elastin, with preferential cleavage at Ala-Xaa.
Trypsin is a serine endoprotease that preferentially cleaves at Arg-Xaa and Lys-Xaa. Trypsin is identified by EC 18.104.22.168.
Chymotrypsin is a serine endopeptidase identified by EC 184.108.40.206.
Pronase (a registered trademark of Calbiochem/Behring, La Jolla, Calif.) is a mixture of various exo-and endoproteases, obtained from Streptomyces. It is able to hydrolyze virtually any protein almost completely to free amino acids.
Dispase is a neutral metalloprotease typically obtained from Bacillus polymyxa.
Bromelin is a cysteine protease that has broad specificity and is identified by EC 184.108.40.206.
Clostripain is a cysteine protease that preferentially cleaves Arg-Xaa and Arg-Pro bonds and is identified by EC 188.8.131.52.
Thermolysin is a metalloendoprotease that preferentially cleaves Xaa-Leu and XaaPhe and is characterized as EC 18.104.22.168.
Subtilisin is a serine endoprotease that catalyzes the hydrolysis of proteins with broad specificity for peptide bonds and is identified by EC 22.214.171.124.
Papain is a cysteine endopeptidase that preferentially hydrolyzes peptide bonds at the carbonyl end of Arg, Lys, Phe residues and is identified by EC 22.214.171.124. Papain also has esterase, thioesterase, transamidase, and transesterase activity.
Chymopapain is an endoprotease with specificity similar to that of papain.
Chymopapain is identified by EC 22.214.171.124.
Fibrinolysin is also known as plasmin, which is a serine protease that converts insoluble fibrin of a blood clot into soluble products. It is formed from plasminogen by proteolysis and preferentially cleaves Lys-Xaa and Arg-Xaa. Fibrinolysin is identified by EC 188.8.131.52.
Cathepsin G is a glycoprotein serine endoprotease with a specificity similar to that of chymotrypsin. This protease is identified by EC 188.8.131.52.
Plasminogen activators include any serine protease that converts plasminogen into plasmin.
PMN leukocyte serine protease includes a serine protease found in PMN leukocytes.
Other proteases include but are not limited to pancreatin and serrathiopeptidase.
Nucleases, Esterases, and Lipases
Examples of nucleases suitable for use in methods of the invention include DNase I and RNase. DNase I (Deoxyribonuclease I) is an enzyme that catalyzes the endonucleolytic cleavage of DNA into 5xe2x80x2-phosphodinucleotide and 5xe2x80x2-phosphooligonuleotide end products. DNase I is identified by EC 18.104.22.168. RNase is any of a group of nuclease enzymes that cleave phosphodiester bonds and chains of RNA.
Examples of esterases suitable for use in methods of the invention include cholesterol esterase.
Examples of lipases suitable for use in methods of the invention include phospholipase. Phospholipase is an enzyme that catalyzes the hydrolysis of a glycerophospholipid. Phospholipases have been subdivided into types A1, A2, B, C, and D.
Calcium Ions
Calcium ions can also be administered to treat prostate cancer. Calcium ions can serve to activate both collagenase and/or PSA. The calcium ions can be provided by, for example, calcium chloride. The concentration of calcium ions is typically between about 1 mM and 50 mM, preferably between about 10 mM and 50 mM, and more preferably about 20 mM.
According to one treatment, activated PSA can degrade or dissolve a prostate tumor and interfere with initial tumor growth and/or angiogenesis. According to another treatment, collagenase can be administered in combination with PSA being activated, and the activated PSA can increase the proteolytic activity of collagenase. Collagenase can, in turn, degrade a prostate tumor. According to another treatment, collagenase can be administered in combination with calcium ions and/or a protease, an enzyme, a protein, a nonionic surfactant, or an antibiotic to treat prostate cancer.
Nonionic Surfactants
Compositions suitable for use in methods of the present invention also can include or be administered in combination with a nonionic surfactant. A surfactant aids in the solubilization and lysis of prostatic cancer tissue. Examples of suitable surfactants include alkylphenylpolyoxethylene surfactants such as Triton(copyright) X-100 (octylphenoxypolyethoxyethanol, which is an octylphenylpolyoxyethylene oxide available from Rohm and Haas, Philadelphia, Pa.) and other polyoxyalkylene-based nonionic surfactants such as Tween(copyright) 20/80 (Atlas Chemical), Genapol X-080/100/150, C-100 (Hoechst AG), Thesit (Destin-Werk GMBH), Brij 35, Lubrol PX (ICI Americas), Pluronic F-127 (Wyandotte Chemicals Corp.), Nonidet P-20/40 (Shell Oil Corp.), Igepal CO-630/710 (GAF), Surfonic N-95 (Jefferson), Tergitol NP-27 (Union Carbide). Other suitable surfactants include the condensation products of ethylene oxide with partial fatty acid esters of sorbitol and sorbitol anhydride, such as, for example, the Tween(copyright) series (Atlas Chemical) wherein the molar ratio of ethylene oxide to alcohol is within the range of about 15:1 to 25:1 with the fatty acid component comprised of laurate, stearate, or oleate (C10-C20).
Other nonionic surfactants that can be employed in the present composition include the ethylene oxide esters of C6-C12 alkyl phenols such as nonylphenoxy polyoxyethylene ether. Particularly useful are the esters prepared by condensing 8 to 12 moles of ethylene oxide with nonylphenol. Commercially available detergents of this type include the Igepal CO series (GAF Corp.).
Additional useful nonionic surfactants can include the condensation products of ethylene oxide with a hydrophobic polyoxyalkylene base such as propylene oxide condensed with propylene glycol. Compounds of this type include the commercially available surfactants Pluronic F-127, Pluronic PX, and Pluronic L-62 (Wyandotte Corp.).
Further useful nonionic surfactants include the condensation products of C8-C22 alkyl alcohols containing 2 to 50 moles of ethylene oxide per mole of alcohol. Detergents of this type include the condensation products of C10-C20 fatty alkyl alcohols containing 3 to 45 moles of ethylene oxide per mole of alcohol. These compounds are commercially available as the Poly-Tergent SLF series (Olin Chemicals) or the Tergitol series (Union Carbide).
The performance of suitable surfactants can be effected by, for example, pH, temperature, ionic strength, and surfactant concentration.
Surfactants are preferably added to the present composition at a concentration of about 0.1% to 10% by volume of the composition. More preferably, the surfactants are present at a concentration of about 0.5% to 5% by volume.
Antibiotics
Compositions suitable for use in methods of the invention also can include or be administered in combination with an antibiotic. Local administration to the prostate, such as by, for example, direct injection of the prostate, is generally a safe, simple, and effective means of introducing the composition into the body. Yet some local administration techniques can be associated with the risk of acquiring bacterial infection that can lead to, for example, fever, bacteriuria, and bacteremia.
Antibiotics usually relieve the symptoms of acute prostatic infections promptly. However, no antimicrobial agent is effective against all pathogenic urinary tract microorganisms. Each has its own spectrum of activity against one or a variety of species. The therapeutic agents for curing bacterial prostatitis preferably are highly lipid soluble; possess basic pKa; show minimal binding to plasma proteins; and are bactericidal against the common gram negative uropathogens.
It is preferred that the present enzyme composition include a suitable antimicrobial agent to prevent or reduce the incidence of bacterial infection that can be associated with the present injection method. Antibiotics used typically provide adequate protection against the commonly encountered bacterial strains of uropathogens including: Escherichia coli, Streptococcus faecalis, Proteus/Pseudomonas spp., and coagulase-positive Staphylococcus. The antibiotic is preferably selected to not substantially inhibit the enzymatic activity of the composition.
The antibiotics of the present claimed composition, preferably gentamicin or trimethoprim/sulfamethoxazole, can be selected from the groups of antibiotics that exhibit the appropriate spectrum of activity against the commonly encountered bacterial strains of uropathogens including: penicillins (penicillin G, penicillin V, benzathine penicillin); amino penicillins (ampicillin, amoxicillin); carboxy penicillins (carbenicillin, piperacillin, mezlocillin); penicillinase resistant penicillins (methicillin, oxacillin, nafcillin); cephalosporins (cephalexin, cephalothin, cefotaxime, cephazolin); aminoglycosides (streptomycin, neomycin, kanamycin, tobramycin, amikacin, netilmicin, sisomicin); tetracyclines (doxycycline, minocycline, tetracycline), polymyxins (polymyxin B and E); sulfonamides (sulfisoxazole, sulfasuxidine); fluoroquinolones(ciprofloxacin,norfloxalin); basic macrolides (erythromycin, oleandomycin); lincomycin; clindamycin; chloramphenicol; nitrofurantoin; and nalidixic acid.
Examples of antibiotics suitable for use in methods of the invention include gentamicin sulfate (Garamicin(copyright), Schering Corp., Kenilworth, N.J.), trimethoprim/sulfamethoxazole (Septrag, Burroughs Wellcome, Research Triangle Park, N.C.), nitroftirantoin, nalidixic acid, tobramycin, amikacin, and netilmicin sulfate.
Preferably gentamicin sulfate or trimethoprim/sulfamethoxazole are administered. The administration of trimethoprim/sulfamethoxazole currently is the drug of first choice in the treatment of bacterial prostatitis. Recent studies have indicated that a single dose of gentamicin, trimethoprim/sulfamethoxazole, or netilmicin sulfate is as effective as longer treatment in the prevention of postoperative bacterial urinary tract infections.
Trimethoprim is a lipid-soluble base with limited binding to plasma proteins, and typically shows prostatic tissue: serum levels of 2:1 to 3:1. Trimethoprim/sulfarnethoxazole (TMP/SMX) produces therapeutic levels in the urine and prostatic secretions with an appropriate antibacterial spectrum of activity. Recommended therapy with TMP/SMX involves dosages of 160 mg TMP and 800 mg SMX, orally twice a day for 30 days. Should TMP/SMX not be tolerated (allergies), gentamicin therapy is recommended.
The antibiotic generally is present in the composition at a concentration of about 0.15 to 150 xcexcg/ml. The preferred antibiotic, gentamicin sulfate, is present in the composition at a concentration of 1.5 to 150 xcexcg/ml, preferably 10 to 25 xcexcg/ml. Alternatively, for those patients who may be allergic to aminoglycosides in general, and gentamicin in particular, the combination drug trimethoprim/ sulfamethoxazole can replace gentamicin as the preferred antibiotic of the present claimed composition. Trimethoprim is preferably present at a concentration of 1 to 10 xcexcg/ml, more preferably 5 to 10 xcexcg/ml. Sulfamethoxazole is preferably present at a concentration of 30 to 105 xcexcg/ml, more preferably 50 to 105 xcexcg/ml.
Preparation and Testing of an Illustrated Composition
The present invention discloses as a preferred embodiment an aqueous composition containing a safe and therapeutically effective concentration of the hydrolytic enzymes collagenase and hyaluronidase, the detergent Triton(copyright) X-100, and the antibiotic gentamicin for local administration. It is preferred that the disclosed composition be prepared as a relatively concentrated solution of hydrolytic enzymes in a relatively small volume.
It is preferable that the claimed composition be provided in a unit dosage form suitable for intraprostatic injection. The composition can be administered to the patient as an injectable dosage of a solution or suspension of the compounds in a physiologically acceptable liquid diluent, such as pyrogen-free saline. For example, vials containing a lyophilisate of the composition can be prepared such that a sterile aliquot of the composition can be reconstituted and withdrawn as a pharmaceutically acceptable aqueous solution for injection into living mammals. A preferred unit dose contains 250 to 250,000 U/ml collagenase; 160 to 160,000 U/ml hyaluronidase; 0.1% to 10% nonionic surfactant; and 0.15 to 150 xcexcg/ml antibiotic. More preferably a unit dose contains 2,500 to 25,000 U/ml of collagenase; 1,600 to 16,000 U/ml of hyaluronidase; 0.5 to 5% nonionic surfactant; and 15 to 150 xcexcg/ml antibiotic.
Compositions of the present invention were prepared. Collagenase (Sigma Chemical Co., St. Louis, Mo.) and hyaluronidase (Boehringer/Mannheim Corp., Indianapolis, Ind.) were obtained as lyophilisates and reconstituted with citrate-buffered saline containing 20 mM to 50 mM CaCl2 (CBSCa) to the desired concentration. Collagenase, obtained from Clostridium hystolyticum, was chromatographically purified and contained small contaminating amounts of the enzymes clostripain, trypsin, and caseinase. Hyaluronidase, obtained from ovine testes, was also purified chromatographically. All enzyme activities were expressed as international units per mg.
The enzymes are stable when stored as lyophilisates at 4xc2x0 C. However, it is advantageous to prevent access of moisture to lyophilized enzymes. For example, cold vials of lyophilized enzyme are typically first be warmed to room temperature before being opened. Dilute reconstituted solutions of enzymes are typically stored at 4xc2x0 C., protected from light, and placed in an ice-bath when working at the bench.
Freshly distilled, deionized, sterile water is preferred for the reconstitution of enzymes and preparation of buffers used for injectable solutions. In the preferred embodiment, the buffer solution used is 0.05 M citrate-buffered saline (CBS, pH 6.7), containing an adequate amount (preferably 0.01 M to 0.05 M, more preferably 0.02 M to 0.05 M) of calcium ions to activate the collagenase. It is recognized that any suitable buffer solution such as Ringers saline or tris-buffered saline can be used. However, the buffer typically contains sufficient calcium ions to activate collagenase, and typically does not contain calcium chelators such as EDTA or other inhibitors of enzyme activity such as cysteine.
A preferred buffer exhibits a physiological pH, which can range from about 6.5 to 7.5, with pH 6.7 to 7.0 preferred. The saline, e.g., sodium chloride, concentration is preferably about 0.1 M to 0.2 M with about 0.15 M to 0.2 M most preferred. Similarly, the concentration of citrate is preferably about 0.02 M to 0.1 M with 0.05 M to 0.1 M most preferred.
A preparation of 0.05 M CBS+20 mM CaCl2 (pH 6.7) is composed of 550 mg sodium citrate, 190 mg NaOH, and 876 mg NaCl dissolved in 100 ml sterile, pyrogen-free, deionized H2O. The solution was adjusted to a pH of 6.7 with 3 ml of 1 N NaOH, and 294 mg CaCl2 was added. The surfactant Triton(copyright) X-100 (Malinckrodt, Paris, Ky.) and the antibiotic gentamicin (Sigma Chemical Co.) were added to obtain appropriate final concentrations. Triton(copyright) X-100 exhibits a density of 1.082 g/ml at 20xc2x0 C. (924 Tl/g at 20xc2x0 C.). Gentamicin is included so as to obtain a final concentration of 150 xcexcg/ml by adding 1.5 ml of a 10 mg/ml (15 mg) sterile solution of the antibiotic to 100 ml of the mixture.
The resulting solution can be purified and sterilized by standard techniques. A solution (5 ml) of the enzymes collagenase and hyaluronidase (0.1% to 10%) in citrate buffered saline (pH 6.7) containing Triton(copyright) X-100 (0.1% to 10%), the antibiotic gentamicin (1.5 to 150 xcexcg/ml), and CaCl2 (20 mM) is prepared in pyrogen-free water, and passed over a 1 ml column (Detoxi-Gel(copyright)) to remove potential endotoxins. The final step in the preparation of a pharmaceutically acceptable solution involves the passage of the composition through a certified sterile, nonpyrogenic microporous polysulfone filter with a pore size of 0.2 micrometer. Low protein binding filter membranes composed of polysulfones show significantly less protein absorption than comparable cellulose acetate/nitrate membrane filters. Filtration through a 0.2 mm sterile filter affords protection against contamination with microorganisms. Additionally, filtration minimizes the risks to patients posed by insoluble particulates or microaggregates.
Reconstitution and preparation of pharmaceutically acceptable solutions for parenteral use in humans is performed routinely in hospital pharmacies as standard practice. Solutions of collagenase/hyaluronidase/Triton(copyright) X-100/gentamicin (CHTG) in 0.05 M citrate-buffered saline containing 20 mM CaCl2 (CBSCa 6.7) ranging in concentration from 0.1% to 10% are stable for 2 weeks when stored at 4xc2x0 C. and remain highly effective in solubilizing human and canine prostatic tissue. The toxicity of the composition is not believed greater than the toxicities of the individual components, which are known.
Collagenase (EC 188.8.131.52) derived from Clostridium hystolyticum was purchased from Sigma Chemical Company (Type XI, Product # C-7657, Lot Numbers: 96F-6801 and 96F-6838; Type XI-S, Product # C-4785, Lot Number: 17F-6814). Lot to lot variations of enzyme activity in terms of collagenase U/mg and levels of contaminating enzymes were observed and ranged from: 1910 to 2450 U/mg collagenase, 0.86 to 1.4 U/mg clostripain, 40 to 85 U/mg caseinase, and 0.05 to 0.52 U/mg trypsin.
One unit of collagenase activity is defined as the amount of collagenase that will release peptides from native collagen, equivalent in ninhydrin color, to 1 micromole of L-leucine in five hours at pH 7.4 at 37xc2x0 C. in the presence of calcium ions. One unit of clostripain will hydrolyze 1 micromole of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) per minute at pH 7.6 at 25xc2x0 C. in the presence of 2.5 mM dithiothreitol. One unit of caseinase (nonspecific protease) will hydrolyze casein to produce color equivalent to 1 micromole (181 Tg) of L-tyrosine in five hours at pH 7.5 and 37xc2x0 C. (color by Folin-Ciocalteu reagent). One unit of trypsin activity will hydrolyze 1 micromole of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) per minute at pH 7.6 and 37xc2x0 C.
The activities of collagenase and any additional enzymes can be determined according to methods known in the art. Examples of collagenase and hyaluronidase assays are disclosed in U.S. Pat. No. 5,116,615 issued to Gokcen et al., incorporated herein by reference.
Compositions are essentially free of endotoxins, which are pyrogenic lipopolysaccharide components of gram-negative bacteria that are known to have potent adverse effects in humans and animals.
Methods of Delivery
One method of the invention is directed to activating PSA in vivo. In one embodiment, PSA can be activated in vivo by, for example, locally administering a therapeutically effective concentration of calcium ions. PSA can be activated in vivo in combination with locally administering a therapeutically effective amount of collagenase, which can be administered in combination with a selected protease, protein, or enzyme, calcium ions, a surfactant, and/or an antibiotic.
Another method of the invention is directed to administering an aqueous parenteral composition suitable for treating prostate cancer. Suitable compositions are described above and include a therapeutically effective amount of collagenase and optionally include one or more of the following: an above-identified selected protease, protein, or enzyme, calcium ions, a surfactant, and an antibiotic. In a preferred embodiment, the composition includes collagenase, hyaluronidase, calcium ions, a surfactant, and an antibiotic. The calcium ions can be supplied by, for example, CaCl2. The surfactant can be, for example, Triton(copyright) X-100, and the antibiotic can be, for example, gentamicin.
The composition is locally administered to the prostate. Local administration includes delivering the composition into or near the prostate and/or cancerous cells or tumor, preferably into the tumor. Local administration also includes surrounding the prostate and/or cancerous tumor with the composition or applying the composition to the surface of the prostate and/or cancerous tumor. For example, the composition can be locally administered by direct intraprostatic injection or by infusion into the prostate. Small volumes, such as, for example, about 5 cc or less, are preferably injected. Larger volumes, such as, for example, greater than about 5 cc, are preferably transfused slowly over, for example, about 15 to 20 minutes.
To verify the accuracy of the administrationxe2x80x94i.e., whether the composition is administered near or into the cancerous cells or tumorxe2x80x94the urologist typically has a good mapping of the prostate from, for example, ultrasonic imaging, which can be obtained during the diagnostic work-up and prostate biopsy.
The composition is typically administered such that local administration of therapeutically effective amounts of the composition result in treating prostate cancer.
Preferably the amount is effective to degrade the prostate tumor, either curing or alleviating prostate cancer.
In one embodiment, the composition is locally administered by intraprostatic injections. Intraprostatic injections are carried out by inserting a long, fine needle into the prostate under digital rectal control and/or ultrasonic guidance. The injections are usually done under local anesthesia, and the injection solution can be diluted with iodocaine. During the injection, the needle can be frequently relocated in order to obtain the best possible distribution of the composition. Several routes of injection are available for the introduction of the disclosed composition to the prostate.
The preferred route of administration is by means of transurethral intraprostatic (intralesional) injection. The transurethral technique is immediately preceded by catheterization. The volume of the composition injected typically varies from 1 to 50 cc.
To optimize the effectiveness of the injected composition, it can be desirable to dilate the prostatic urethra with an inflatable balloon. The cystoscopically inserted balloon inhibits the immediate egress of the injected enzyme solution through the porous duct system that empties into the urethra. The advantage of this route of injection is that the method allows for direct cystoscopic visualization of the nodular areas of pathology and for the placement of a high concentration of the composition at the desired location without the risk of metabolic inactivation. The pain and discomfort experienced by patients during direct injection of the prostate typically are minimal and comparable to intramuscular injections.
Alternatively, the transperineal or transrectal routes of prostatic injection can be used. The transperineal route of injection involves the placement of 22 gxc3x9720 cm aspiration biopsy needle through the perineum into the prostate guided by ultrasound and/or digital palpation. Again, 1 to 50 cc of the disclosed composition is typically injected into each lateral lobe of the prostate. The injections are generally done under local anesthesia. During injection, the needle is frequently relocated to obtain the best possible distribution of the composition. The position of the needle can be guided by ultrasound while kept under constant digital rectal control. The transperineal route of injection can be a better alternative than either the transurethral or transrectal routes in terms of reducing potential complications due to postinjection bacterial infection.
To reduce the incidence of bacterial infection that can be associated with transperineal intraprostatic injection, aseptic injection techniques are recommended and are well known to those skilled in the art. Any one of a variety of standard bactericidal preparations such as Phiso-Hex(copyright), Betadine(copyright), povidone-iodine, or chlorhexidine applied to the skin of the perineum provides adequate pre-injection antibacterial protection. With sterile urine, adequate skin preparation, and sterile technique, the entire procedure should have a low rate of infectious complication.
The transrectal route allows needle introduction through the rectal wall and injection of the prostate while performing digital rectal palpation. Injection via the transrectal route is performed with a slightly curved 22 gxc3x9720 cm flexible aspiration biopsy needle. The use of a Franzen needle guide (Precision Dynamics, San Fernando, Calif.) allows the needle to be safely directed into a suspected lesion under ultrasonic and/or tactile guidance techniques. The sterilized prostate needle guide is placed on a gloved index finger. A finger cot is placed over the needle guide. The index finger and needle guide are inserted into the rectum and suspected lesions of the prostate are palpated. The needle is inserted through the guide and advanced into the tissue. Approximately 1 to 50 cc of the solution can be injected into the lateral lobes of the prostate. In order to inject sufficient material, the needle can be moved back and forth three to five times. An anesthetic jelly can be applied before injection to reduce pain during needle puncture.
Upon injection, the prostatic lobe swells, increases in size, and becomes turgid. Injected fluid forced through the veins at the site of injection can induce wide spread venospasm associated with microinfarcts. Acute urinary retention can occur in the immediate post injection period. Fluid injected into the prostate fills the alveoli of the gland at the site of injection and can rupture through the walls of adjacent alveoli entering the prostatic urethra via the alveolar ducts. As much as ⅓ to xc2xd of the injected fluid can ultimately reach the prostatic urethra.
A bolus injection of more than 5 cc of the composition into the body of the in situ prostate can result in reflexive smooth muscle contraction causing the therapeutic enzyme solution to be rapidly emptied through the porous ducts, away from the target tissue, and into the urethra. The force of the injection can cause rupture of prostatic tissue at the site of injection. The injected fluid can gain access to the ducts of the glandular alveolar system and completely fill the gland. Once the gland is filled, the fluid takes the path of least resistance and flows to the urethra.
Injection fluid can gain access to the prostatic circulation and be responsible for scattered minute areas of infarction. Enzyme induced thrombophlebitis of the veins can be responsible for the appearance of widespread hemorrhagic infarctions. About ⅕ of the prostatic injection fluid can enter the general circulation as demonstrated by experiments involving excretion of methylene blue by the kidneys. The subcapsular and periurethral zones of the prostate are more vascular in nature and can enhance the flow of the fluid into the blood stream. Injections of India ink carbon particles into the prostate resulted in no gross or microscopically detectable particles in the lymphatics of the pelvis. Prostatic injection fluid can also reach the surface of the prostate and periprostatic tissue through the point of needle entry.
Radiographs taken immediately after intraprostatic injection of radio-opaque microemulsions of barium sulfate have shown fluid leakage beneath the prostatic capsule and escape into the bladder. Occasionally, the fluid has been seen to leak outside the capsule as well.
The injected lobule of the prostate undergoes necrosis mainly due to the enzymatic action of the injected composition and partly due to the shear force of the fluid injected under pressure. Part of the fluid that leaks back through the point of needle entry can cause thrombosis of surface vessels and be responsible for adhesions with adjacent viscera. The quantity of fluid that leaks back depends upon the local pressure and is likely to increase with the force of the injection. Injected fluid passing through the periurethral veins can cause their inflammation and thrombosis, which in turn can result in necrosis and sloughing of the urethral epithelium.
Urethral and periurethral solubilization is expected to occur around the entire circumference of the prostatic urethra which can lead to denudation of the urethral epithelium. Histopathologic changes associated with injection of the tissue solubilizing enzymes include those due to fluid escape along ducts of the gland resulting in damage to the ducts and surrounding alveoli. The fluid that leaks back through the point of needle entry can affect the capsular vessels and smooth muscle fibromuscular stroma situated there.
Direct localized injection of the prostate results in a high concentration of therapeutic enzymes at the very focus of the problem without the risk of metabolic inactivation. However, intraprostatic injections during acute exacerbations of infection are not recommended because of the danger of general dissemination of the infection and possible septicemia. Hematuria and hemospermia can be present for some weeks following the injection.
Depending upon the patient being treated, the therapeutically effective dose of the composition administered can range from 1 cc to 50 cc preferably containing 160 to 160,000 U/ml hyaluronidase; 250 to 250,000 U/ml collagenase; 0.1 to 10% nonionic surfactant preferably Triton(copyright) X-100; and 0.15 to 150 xcexcg/ml antibiotic, preferably gentamicin. More preferably, a dose includes 2,500 to 25,000 U/ml collagenase; 1,600 to 16,000 U/ml hyaluronidase; 0.5 to 5% nonionic surfactant, preferably Triton(copyright) X-100; and 15 to 150 xcexcg/ml antibiotic, preferably gentamicin. These dosage ranges represent quantities of the various components of the composition that are estimated to be therapeutically effective for treating prostate cancer. But the dose of the composition can vary depending on the age of the patient, nature and severity of disease, potency of the composition, and route of administration. Treatment regimens encompassed by a preferred embodiment of the present invention employ the intraprostatic injection of safe and effective amounts of the preferred composition to cause the regression of prostate cancer. The injections can be administered in daily, weekly, or monthly injection protocols until the therapeutically desired result is obtained.
In some embodiments, to determine the appropriate dosage, the size of the tumor is estimated because a larger tumor can require a higher concentration of the composition suitable for use in methods of the invention. For example, for a tumor smaller than 7 g, a single bolus dose of the composition is typically administered. A bolus dose can be 5 cc and preferably contains 250-250,000 U/ml collagenase, 160-160,000 U/ml hyaluronidase, 0.1% to 10% nonionic surfactant, 0.15 to 150 xcexcg/ml antibiotic, and 20 mM to 50 mM CaCl2 in citrate buffer (pH 6.7). The bolus dose is preferably injected slowly into the tumor, for example, over several minutes. This injection can be followed by additional injections if necessary. For example, this treatment can be administered again in, for example, a week or a month or can be administered repeatedly, for example, weekly or monthly.
For a tumor between 7 and 15 g, a first bolus dose is typically followed by a larger dose of the composition. For example, the 5 cc bolus dose described above is first administered. Then, a second dose of the same composition in 25 to 45 cc is slowly administered over, for example, about 15 to 20 minutes. These injections can be succeeded by still another dose if necessary, but a total volume of 50 cc for all doses is typically not exceeded. For example, this treatment can be administered again in, for example, a week or a month or can be administered repeatedly, for example, weekly or monthly.
For a tumor larger than 15 g, a dose of up to about 50 cc can be administered by first administering a bolus dose followed by a larger dose as described above.
Other Methods of Delivery
If problems are encountered that limit or inhibit the therapeutic effects of the present composition, alternative means of delivery can be used. For instance, the effectiveness of enzyme therapy can be limited by the short circulating half-lives of exogenously administered enzymes, by the development of immunological responses to foreign protein, by inhibition from antiproteinase effectors (I-1-antitrypsin, I-2-macroglobulin), or by the inability to specifically target the enzymes to nodular areas of pathology.
A number of different carrier systems can be utilized to carry the enzymatic composition to the desired site in the prostate. In general, a suitable carrier guides the therapeutic agent to its target without loss of specificity or reactivity. The carrier is preferably capable of linkage with the therapeutic enzymes and remaining as a complex until delivery is completed. The carrier preferably avoids triggering the immune defense mechanisms resulting in biodegradation or inactivation of the present composition.
The composition can be administered as a depot formulation that permits sustained release, prevents access to general circulation, arid increases the prostate-specific localization of the composition. Such a formulation can be provided as a slow-release implant or can be microencapsulated or attached to a biodegradable polymer or a prostate-specific immunoglobulin.
The use of antibodies as an enzyme composition carrier system can be desirable. The use of antibodies as carrier systems for the delivery of therapeutic agents to specific tissues exploits the antibody""s unique ability to recognize and bind to targeted antigens. Also, antibodies carrying therapeutic reagents can be more effectively localized in tissues that are highly vascular in nature or are undergoing neovascularization. Additionally, cocktails of immuno-enzyme conjugates recognizing different cell types with different specificities can be useful.
Tissue-specific monoclonal antibodies can be produced that better define the antigenic cellular targets to be localized. The use of F(ab) or F(abxe2x80x2)2 fragments can improve localization properties. Antibodies containing the F(c) fragment tend to be specifically localized over a longer time period than F(ab) or F(abxe2x80x2)2 fragments that exhibit accelerated clearance mechanisms. The recent development of human-mouse (chimeric) monoclonal antibodies can have therapeutic applications and provide advantages over conventional monoclonal antibodies of murine derivation. Human-mouse antibodies exhibit a wide range and high degree of specificity. Chimeric antibodies are less likely than conventional mouse monoclonal antibodies to elicit an immune reaction when injected into humans. The preparation and use of human monoclonal antibodies as carriers can further reduce the degree of immune response in the recipient to the introduction of foreign proteins. The antiglobulin response can also be controlled by such factors as method of antibody preparation, dosage, and route of injection.
The purpose of immuno-targeted enzyme therapy is to deliver an effective concentration of enzymes to a tissue specific site of activity, reduce toxicity to nearby normal tissues, and thereby increase the therapeutic index. Enzymes can be coupled to monoclonal antibodies that bind the enzyme covalently yet do not affect the enzyme""s catalytic activity. Enzymes that are coupled to tissue specific monoclonal antibodies can be able to achieve a higher degree of specific localization in the targeted tissue than native enzymes while maintaining their proteolytic activity.
Other specific localization concepts include zymogen-antibody conjugates (trypsinogen) or enzyme-antibody conjugates (collagenase, hyaluronidase, elastase, DNase) that retain both enzyme and antibody activity. Enzymes can be encapsulated in lipoprotein, red blood cell (RBC) ghosts, polylactic acid, and other biodegradable membranes or synthetic microcapsules containing prostate-specific antibodies to increase and maintain specific targeting, localization, and activity of the solubilizing proteases in prostatic tissue.
The administration of collagenase and hyaluronidase can have immunologic consequences as repeated injections can result in the development of antibody titers and the accompanying risk of anaphylaxis or other less serious hypersensitivity reactions. Additionally, the presence of specific antibodies to collagenase or hyaluronidase can inhibit enzyme activity. Potential immunologic problems could occur if the active enzyme is recognized as foreign by the recipient""s immune system. Antibodies can be produced against the enzymes and inactivate or precipitate the enzyme. Use of enzymes of human origin or those produced by recombinant techniques can minimize these potential immunologic complications.
Strategies that avoid the immune surveillance system involve methods of entrapment of enzyme preparations in biodegradable vesicles that protect the enzyme activity yet facilitate specific delivery. Targeting to specific sites of cellular pathology can be accomplished by attaching tissue specific proteins (monoclonal antibodies) to these vesicles. Enzymes can also be encapsulated in liposomes or other biodegradable microcapsules and subsequently attached to tissue-specific monoclonal antibodies for specific localization purposes.
Liposomes are small spheres of concentric phospholipid bilayers containing an aqueous phase that have been shown to be useful as carrier systems. Current liposome preparation techniques permit the incorporation of a variety of drugs, hormones, or enzymes into either phase. Monoclonal antibodies can be incorporated into the outer layers of liposomes and provide increased specificity of delivery for the liposome-contained therapeutic agent.
The entrapment of enzymes in synthetic microcapsules or biodegradable vesicles can provide a valuable method of specific delivery in addition to protecting the enzyme from physiologic inactivation and preventing immune complications. Various forms of membrane encapsulation techniques are available for the entrapment of enzymes including: erythrocyte ghosts; synthetic polymeric microcapsules; and lipid vesicles (liposomes) composed of cholesterol, lecithin, and phosphatidic acid. Use of the recipient""s own erythrocytes to deliver active enzyme can avoid the potential immunologic and physiologic problems resulting from enzyme administration in synthetic carriers (e.g., liposomes and microcapsules).
The covalent attachment of polyethylene glycol (PEG) to enzymes renders these proteins nonimmunogenic, can extend their circulating half-life, provides a means of escape from inhibition by naturally occurring enzyme inhibitors, and can result in enhanced enzyme activity with decreased autodigestion. The attachment of PEG to proteins is simple and yields homogeneous reaction products that can be purified by ultrafiltration.

Method and composition for treating prostate cancer